Vitamin D, acquired either from dietary sources or via ultraviolet irradiation of 7-dehydrocholesterol in the epidermis, is metabolized to its hormonal form. The keratinocytes of the skin are unique in being not only the primary source of vitamin D for the body, but also possessing the enzymatic machinery to metabolize vitamin D to active metabolites. Many functions of the skin are regulated by vitamin D and/or its receptor: these include inhibition of proliferation, stimulation of differentiation including formation of the permeability barrier, promotion of innate immunity, regulation of the hair follicle cycle, and suppression of tumors.
When exposed to ultraviolet radiation, cells in the epidermis convert a cholesterol related steroid to vitamin D or cholecalciferol. Vitamin D is essential for proper development of the bones. The ultraviolet radiation necessary for vitamin D synthesis (specifically, UV-B) only reaches the Earth's surface in much abundance for a few hours a day when the sun is high. Much less of it reaches the Earth's surface at high latitudes than at low latitudes, and very little reaches the Earth's surface on cloudy days or during the winter. Even so, the average fair-skinned person can make and store several days' worth of vitamin D with just one hour's exposure to the midday sun. Dark-skinned people living at high latitudes are much more likely to suffer from vitamin D deficiency than are light-skinned people.
The most important source of Vitamin D is through the action of sun on cholesterol on the skin. Vitamin D modulates T-cell responses and has anti-inflammatory properties, and boosts innate immune responses by induction of the human gene for cathelicidin. Cathelicidin's and defensins are small peptides with amphipathic structures that allow them to disrupt the integrity of the pathogen cell membrane, resulting in its death. Most immune cells or those epithelial cells that are in contact with the environment express these proteins. Deficiency in these peptides results in increased susceptibility to infection.
Vitamin D enters the circulation and is transported to the liver, where it is hydroxylated to form 25-hydroxyvitamin D3 (calcidiol; the major circulating form of vitamin D). In the kidneys, the 25-hydroxyvitamin D3-1-hydroxylase enzyme catalyzes a second hydroxylation of 25-hydroxyvitamin D, resulting in the formation of 1,25-dihydroxyvitamin D3 (calcitriol, 1alpha, 25-dihydroxyvitamin D]—the most potent form of vitamin D. Most of the physiological effects of vitamin D in the body are related to the activity of 1,25-dihydroxyvitamin D. Keratinocytes in the epidermis possess hydroxylase enzymes that locally convert vitamin D to 1,25 dihydroxyvitamin D3, (Bikle, et al. 1986, Biochemistry. 25 (7): 1545-15480) the form that regulates epidermal proliferation and differentiation. In skin, the vitamin D receptor (VDR) appears to have other roles that are independent of its association with 1,25 dihydroxyvitamin D3. For instance, the VDR is important in regulating the growth cycle of mature hair follicles. (Bikle, et al., J. Bone. Miner. Metab, (2010) 28:117-130). Certain mutations in the VDR lead to misregulated gene expression resulting in aberrant hair follicle cycling and alopecia (hair loss) in mice (28, 29) and in humans (30). The VDR also functions as a tumor suppressor in skin. The VDR is one of several factors that control these two diverse roles. Moreover, 1,25-dihydroxyvitamin D3 is a potent immune modulator in skin.
Functions in Healthy Skin
Photo protection photo damage refers to skin damage induced by ultraviolet (UV) light. Depending on the dose, UV light can lead to DNA damage, inflammatory responses, skin cell apoptosis (programmed cell death), skin aging, and skin cancer. Some studies, mainly in vitro (cell culture) studies (Dixon, K M, et al. 2005, J Steroid Biochem Mol Biol, 97(1-2):137-143) and mouse studies where 1,25-dihydroxyvitamin D3 was topically applied to skin before or immediately following irradiation, have found that vitamin D exhibits photo protective effects (Gupta, et al., 2007, J Invest Dermatol. 127(3):707-715). Documented effects in skin cells include decreased DNA damage, reduced apoptosis, increased cell survival, and decreased erythema. The mechanisms for such effects are not known, but one mouse study found that 1,25-dihydroxyvitamin D3 induced expression of metallothionein (a protein that protects against free radicals and oxidative damage) in the stratum basale. It has also been postulated that nongenomic actions of vitamin D contribute to the photo protection; such effects of vitamin D involve cell-signaling cascades that open calcium channels. 1,25-dihydroxyvitamin D3 regulates the expression of cathelicidin (LL-37/hCAP18) (40, 41), an antimicrobial protein that appears to mediate innate immunity in skin by promoting wound healing and tissue repair. One human study found that cathelicidin expression is up regulated during early stages of normal wound healing (Gombart A F, Faseb J., 2005, 19(9):1067-1077). Other studies have shown that cathelicidin modulates inflammation in skin Kratz, G, et al., 2003, J Invest Dermatol. 120(3):379-389), induces angiogenesis (Kupatt C, et al., J Clin Invest., 2003, 111(11):1665-1672), and improves re-epithelialization (the process of restoring the epidermal barrier to re-establish a functional barrier that protects underlying cells from environmental exposures). The active form of vitamin D and its analogs have been shown to up regulate cathelicidin expression in cultured keratinocytes (Sthale M, 2005, J Invest Dermatol., 124(5):1080-1082).
Diseases such as rosacea may require lower levels of vitamin D or even locally active serine proteases inhibitors and vitamin D antagonists to prevent harm. In rosacea, patients might benefit from therapies blocking cathelicidin expression and processing. Polymorphisms in the vitamin D receptor gene have been described in patients with severe rosacea indicating that vitamin D3 signaling is involved in pathogenesis (Jansen T, et. al, J Dermatol 2004; 31:244-246.). Blocking cathelicidin expression by targeting the vitamin D3 pathway might represent a novel therapeutic approach in rosacea. As an example, vitamin D3 analogues without intrinsic activity at the vitamin D receptor have been shown to inhibit 1,25D3-induced cathelicidin in keratinocytes in vitro (Liu P. T, et. al, Science 2006; 311:1770-1773).
In psoriasis, blocking cathelicidin peptide could break the vicious cycle of increased LL-37 expression, dendritic cell activation and cutaneous inflammation. Again strategies to decrease cathelicidin in keratinocytes could target vitamin D3 signaling. Paradoxically, for a long time vitamin D3 analogues have been used in the therapy of psoriasis. Vitamin D3 analogues bind to and activate the vitamin D receptor and should therefore increase cathelicidin in keratinocytes presumably worsening inflammation in psoriasis. However, the opposite is true: vitamin D analogues resemble one of the pillars of topical psoriasis treatment. They ameliorate cutaneous inflammation and reverse morphological changes within skin lesions. (Lebwohl M, et. al, J Am Acad Dermatol 2004; 50:416-430). Understanding the molecular effects of vitamin D3 analogues on cutaneous innate immune function will eventually also lead to better treatment. In summary, influencing cathelicidin expression via vita-min D3 signaling might offer a new treatment angle in the therapy of very common skin diseases. However, until the ‘sunshine vitamin’ can be targeted additional experimental work and clinical studies have to be performed to prove its safety and benefits. Overall, current data overwhelmingly support the importance of AMPs to healthy human skin but the key steps to put this information to therapeutic use remain to be done.
Eczema (Bjorn Hartmann, et. al, Journal of Investigative Dermatology (2012), Volume 132) is a chronic inflammatory skin disease that has reached nearly epidemic proportions in childhood. Moreover, it is a difficult disease to control and, with its onset in childhood, is often the first manifestation of atrophy. The clinical features of eczema include itchy red skin accompanied by dryness and lichenification. In the past, treatment options consisted primarily of avoidance of soap and water. These options have considerably improved with both non-pharmacologic and pharmacologic approaches. However, eczema is still a treatment challenge. Part of the problem in developing new treatment options has been the relative failure in translating basic science information into clinical application. It is hoped that the newer biologics will help bridge this gap and lead to greater success rates.
Atopic dermatitis (AD) is a common chronic inflammatory skin disease that has increased in prevalence over the last several decades in industrialized countries. AD is a multifactorial, heterogeneous disease with a variety of defects in the immune system, in antimicrobial defense mechanisms and epidermal barrier integrity, which collectively contribute to the risk and severity of AD development. (J Innate Immun 2011; 3:131-141).
Topical corticosteroids have been the gold standard for the treatment of atopic dermatitis for many decades. The emergence of the immuno-modulatory drugs Tacrolimus and Picrolimus represented the first major advance in the treatment of this disease in 40 years. Numerous other therapeutic modalities have been studied and whereas some have been found to have beneficial effects, none have exceeded the efficacy of topical corticosteroids. Less severe forms of eczema are generally treated successfully with topical steroids or immuno-modulatory drugs, however,
Steroid-resistant eczema presents a problem because most of the other adjunctive treatments do not completely resolve the condition.
Warts are a benign proliferation of the skin and mucosa caused by infection with human papillomavirus (HPV). HPV is ubiquitous, and renal transplant recipients (RTRs) may never totally clear HPV infections, which are the most frequently recurring infections. This infection is important because of its link to the development of certain skin cancers, in particular, squamous cell carcinoma. Regular surveillance, sun avoidance, and patient education are important aspects of the management strategy. Warts are usually treated by traditional destructive modalities such as cryotherapy with liquid nitrogen, local injection of bleomycin, electrocoagulation, topical application of glutaraldehyde, and local and systemic interferon-β therapy [S. Gibbs, et. al, British Medical Journal, vol. 325, no. 7362, pp. 461-464, 2002. However, the tolerance of patients to these treatment modalities is poor, because they often cause pain, especially in children, and sometimes scarring or pigmentation after treatment. No treatment has been uniformly effective, and warts are often refractory, especially in immuno-compromised patients where their quality of life is threatened. Researchers have reported an RTR with a right index finger wart, which was successfully treated with a topical activated vitamin D. (Luciano Moscarelli, et. al, Case Reports in Transplantation Volume 2011, Article ID 368623).
Hair loss (alopecia) is a much-feared side effect of many chemotherapy protocols and is one of the most psychological devastating aspects of cancer therapy. So far, no satisfactory strategy for suppressing chemotherapy-induced alopecia is at hand. During the last decade, some progress in understanding molecular mechanisms of chemotherapy-induced hair loss has been achieved using rodent models. However, the pathobiology of the response of human hair follicle to chemotherapy remains largely unknown. (Vladimir A Botchkarev, Journal of Investigative Dermatology Symposium Proceedings (2003) 8, 72-75).
Androgenetic alopecia is the most common hair loss disorder in men and is largely determined by genetic factors and the peripheral action of androgens. Others mechanisms such as chronic inflammation and several hormones or vitamins like aldosterone, insulin or vitamin D have been implicated in the pathogenesis of Androgenetic alopecia. The diagnosis of Androgenetic alopecia is made by clinical history and clinical examination. Minoxidil and finasteride are the main drugs approved for the treatment of Androgenetic alopecia. Androgenetic alopecia has been associated with cardiovascular risk factors and benign prostatic hyperplasia. Alopecia is a feature of vitamin D receptor (VDR) mutations in humans and in VDR null mice. This alopecia results from an inability to initiate the anagen phase of the hair cycle after follicle morphogenesis is complete.
Thus, once the initial hair is shed it does not regrow. VDR expression in the epidermal component of the hair follicle, the keratinocyte, is critical for maintenance of the hair cycle. To determine which functional domains of the VDR are required for hair cycling, mutant VDR transgenes were targeted to the keratinocytes of VDR null mice. Keratinocyte-specific expression of a VDR transgene with a mutation in the hormone binding domain that abolishes ligand binding restores normal hair cycling in VDR null mice, whereas a VDR transgene with a mutation in the activation function 2 domain that impairs nuclear receptor co-activator recruitment results in a partial rescue. Mutations in the nuclear receptor co-repressor Hairless are also associated with alopecia in humans and mice. Hairless binds the VDR, resulting in transcriptional repression. Neither VDR mutation affects Hairless interactions or its ability to repress transcription. These studies demonstrate that the effects of the VDR on the hair follicle are ligand independent and point to novel molecular and cellular actions of this nuclear receptor (Kristi Skorija, et. al, Molecular Endocrinology 19: 855-862, 2005).
Previous reports have described the effects of vitamin D on hair follicles. Topical pretreatment of VD3 enhanced hair regrowth in a mouse model of chemotherapy-induced alopecia [Paus R, et. al, Cancer Res 1996; 56:4438-4443). Nuclear vitamin D receptor (VDR)-null-mutant mice were reported to develop alopecia and poor whiskers, although they had normal hair until weaning after birth [Li Y C, et. al, Proc Natl Accad Sci USA 1997; 94: 9831-9835). This suggests that such mice can develop a normal first coat of hair but cannot regulate postnatal hair cycles. In humans, mutations in the VDR coding gene are known to cause hereditary vitamin D resistant rickets with alopecia [Miller J, et. al, J Invest Dermatol 2001; 117:612-617: Zhou Y, et. al, J Bone Miner Res 2009; 24: 643-651]. Furthermore, it has been shown that VDR maintains hair follicle homeostasis that is ligand-independent and suggest that recruitment of novel nuclear receptor co-modulators by the VDR is required for maintenance of hair follicle homeostasis (K. Skorija, et. al, Mol. Endocrinol. 19 (4) (2005) 855-862).
A phase I trial of 14 patients failed to show efficacy for topical calcitriol in the prevention of chemotherapy-induced alopecia (Hidalgo M, et. al, et. al: Anticancer Drugs 10:393-395, 1999 A Phase I trial of topical topitriol (calcitriol, 1, 25-dihydroxyvitamin D3) to prevent chemotherapy-induced alopecia. It has been suggested vitamin D3 resistance may also play a role in alopecia. (Hochberg Z, et. al, Am J Med 77:805-811, 1984).
The Global Burden of Disease (GBD) Study 2010 ((Roderick J Hay, ET. AL, Journal of Investigative Dermatology (28 Oct. 2013) estimated the GBD attributable to 15 categories of skin disease from 1990 to 2010 for 187 countries. At the global level, skin conditions were the fourth leading cause of nonfatal disease burden. Using more data than has been used previously, the burden due to these diseases is enormous in both high- and low-income countries. These results argue strongly to include skin disease prevention and treatment in future global health strategies as a matter of urgency.
Circulating 1,25 dihydroxy D3, bound by DBP (D binding protein), can be delivered systemically to vitamin D target cells that retain the hormone through expression of the nuclear vitamin D receptor (VDR). Intestinal epithelial cells and osteoblasts represent primary sites of VDR (Vitamin D Receptor) expression, where the receptor mediates the actions of 1,25 dihydroxy D3 to promote intestinal calcium and phosphate absorption, and to remodel skeletal mineral, respectively (Haussler, et al. 2013, Calcif Tissue Int, 92:77-98). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a hetero dimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either co-activators or co-repressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. The disease targets, envisioned for vitamin D analogs, appropriately, include osteoporosis by bone-mineral mobilization, secondary hyperparathyroidism to reduce PTH gene transcription and blocking chief cell hyperplasia, autoimmune diseases such as psoriasis and asthma, organ-transplant rejection, benign prostate hyperplasia, involuntary bladder control, blood pressure control by suppressing renin biosynthesis, type 1 diabetes and insulin secretion by affecting pancreatic cell function, anti-inflammatory events via cyclooxygenase-2 (COX-2) inhibition, and cancer via the established anti proliferative and pro differentiating effects on a variety of cell lines, such as breast, prostate and colon. (Pike, et al., 2012, Rev Endocr Metab Disord, 13:45-55).
In this fashion, 1,25 dihydroxy D3 elicits its two major functions of preventing rickets in children and osteomalacia in adults, as well as strengthening bone via remodeling. Thus, although vitamin D has no direct role in bone calcification it is responsible for supplying adequate amounts of calcium and phosphorus minerals.
Extra renal 1,25 dihydroxy Vitamin D3 can also be produced locally in a number of cell types that express VDR, notably skin, cells of the immune system, colon, pancreas, and the vasculature. The significance of local effects of 1,25 dihydroxy D3-VDR is not defined fully, but it appears that vitamin D, likely cooperating with other regulators, exerts immuno regulation, antimicrobial defense, xenobiotic detoxification, anti-cancer actions, control of insulin secretion and cardiovascular benefits. (Hussler, et al. 2013, Calcif Tissue Int, 92:77-98).
1,25-dihydroxycholecalciferol, i.e., calcitriol, the biologically active form of vitamin D is a secosteroid that acts through binding to the VDR inside cells. VDRs have been suggested to reside in the cytoplasm and in the nucleus without hormone in an unbound state. The VDR binds several forms of cholecalciferol; however, its affinity for 1,25-dihydroxycholecalciferol is roughly 1,000 times that of 25-hydroxycholecalciferol. (Hewisson, et al., 2012, Plos One, Volume 7, Issue 1, e30773).
Calcitriol, the active hormonal form of vitamin D, also acts through the VDR to regulate important functions, such as cellular proliferation and differentiation and immune functions. Calcitriol has biphasic effects on cell growth, where physiological doses stimulate cell proliferation, and high pharmacological doses inhibit cell growth. Calcitriol and its derivatives are thought to have utility in the treatment of cancers by retarding tumor growth, inducing apoptosis, and stimulating the differentiation of malignant cells. Current calcitriol derivatives are administered in large dosages to inhibit cancer growth. Unfortunately, such large dosages result in toxic levels of serum calcium.
Further, the therapeutic possibilities of 1,25-dihydroxycholecalciferol are severely limited by the potent effect of this hormone on calcium metabolism, since serious side effects due to hypercalcemia will result from the high doses necessary to obtain a therapeutic effect on, for example, psoriasis, cancer or immunological disorders. To inhibit cell growth, current methodologies utilize combinations of vitamin D derivatives and therapies that specifically alleviate calcemic toxicities incurred by such high pharmacological dosages.
Vitamin D and its analogues, while potentially useful in retarding abnormal cellular proliferation or tumor growth, have the disadvantage of being potent calcemic agents that cause elevated blood calcium levels by stimulating intestinal calcium absorption and bone calcium resorption. Accordingly there is a need for a selective molecular modifications of vitamin D to balance the potential function as a nuclear receptor agonist, antagonist or reverse agonist, and at the same time maintain tissue specificity and sufficient metabolic stability with a constant look out for hypercalcemia and hypophoosphatemia. Therefore, the current focus is directed toward new vitamin D derivatives or analogs with weak calcemic effects and a wide therapeutic window. This and other objects and advantages, as well as additional inventive features, will be apparent from the detailed description provided herein to one of skill in the art.
Compounds which have VDR like activity are known in the art, and are described in numerous United States patents and in scientific publications as agonists and as antagonists; Cited patents include as Agonist (U.S. Pat. No. 6,689,922, U.S. Pat. No. 7,101,865, U.S. Pat. No. 7,595,345, U.S. Pat. No. 7,566,803, U.S. Pat. No. 7,659,296, U.S. Pat. No. 7,750,184) and as Antagonists U.S. patent application Ser. No. 10/481,052, U.S. Pat. No. 7,361,664, U.S. Pat. No. 7,915,242, U.S. patent application Ser. No. 12/266,513, U.S. patent application Ser. No. 10/774,843). It is generally known and accepted in the art that VDR like activity is useful for treating mammals, including humans, to cure or alleviate the symptoms associated with numerous diseases and conditions.
VDR ligands (vitamin D and its derivatives) are known to have broad activities, including effects on cell proliferation and differentiation, in a variety of biological systems. This activity has made Vitamin D derivatives useful in the treatment of a variety of diseases, including dermatological disorders and cancers. The prior art has developed a large number of chemical compounds that have Vitamin D-like biological activity, and voluminous patent and chemical literature exists describing such compounds. (Schrager, et al. 2009, JABFM, 9, Vol. 22, No. 6).
The importance of VDR has been shown by Ramagopalan et al. (2010: Genome Research, 20:1351) by isolating fragments of genomic DNA bound to the VDR before and after treatment of cells with calcitriol, and then sequenced the DNA fragments. By mapping the sequences back to the genome, they identified more than 2,700 sites of VDR binding, a number that shows just how important vitamin D is to humans, and the wide variety of biological pathways that vitamin D plays a role in.
While the discovery of agonist or even “super agonist” activity is known, ligands that selectively stabilize an antagonistic conformation of the VDR LBD within the VDR-RXR-DVRE construct, to prevent induction of transactivation, are also of potential therapeutic value. The degree of affinity of a vitamin-D analog to the VDR appears to be of lesser importance than its alignment with specific contact sites in the ligand binding domain to produce different VDR conformations with modified transcriptional consequences. There are a reported 3000 vitamin D related compounds that have already been synthesized, with the goal to minimize or eliminate hyper calcemic side effects while maintaining sustained plasma levels, the desired transactivation potencies and cell specificities. Many vitamin D analogs that circumscribe the binding affinity and determine its function as agonist or antagonist, the disease modifying potential and, eventually, the inherent clinical value.
Unfortunately, compounds having Vitamin D like activity (e.g.; calcitriol) also cause a number of undesired side effects at therapeutic dose levels, including hypercalcemia. These side effects limit the acceptability and utility of vitamin D for treating diseases. There are no known inverse agonists of VDR and Metadichol® is the first of its kind.